August 2007 1417 with OVA (50 m g, group 1) or with OVA (50 m g)ϩbiﬂorin (1 mg/animal or 40 mg/kg, group 2). The mice were bled from the retro-orbital plexus to obtain sera prior to immu- nization and at 7, 14 and 21 d after. Measurement of OVA-Speciﬁc Antibody OVA-speciﬁcFig. 1. Chemical Structure of Biﬂorin (6,9-Dimethyl-3-(4-methyl-3-pen- total Ig antibodies in serum were detected by enzyme-linkedtenyl)naphtha[1,8-bc]-pyran-7,8-dione) immunosorbent assay (ELISA). Puriﬁed OVA (50 m g protein) diluted in saline solution, at a ﬁnal volume of 100 m l, were Assay of Antitumor Activity A total number of 140 used to coat the 96-well plates. The plates were incubated atSwiss mice (female, 20—30 g) obtained from the central ani- 37 °C for 1 h and washed three times with 0.05% PBS-tween.mal house of the Universidade Federal do Ceará, Brazil, were The plates were blocked with 5% nonfat milk in 10 mMused. Animals were housed in cages with free access to food potassium phosphate buffer, pH 7.2, with 0.9% NaCl (PBS)and water. All animals were kept under a 12 h : 12 h light– for 2 h at 37 °C, washed once, and 100 m l of the appropriatedark cycle (lights on at 6:00 a.m.). Sarcoma 180 and Ehrlich sera diluted in PBS (1 : 10 to 1 : 1280) was added and reincu-carcinoma tumor cells were maintained in the peritoneal cav- bated for 2 h at 37 °C. The plates were washed again threeities of the mice. times with 0.05% PBS-tween and treated with peroxidase- Ten-day-old sarcoma 180 or Ehrlich carcinoma ascites conjugated rabbit antimouse total immunoglobulinstumor cells were removed from the peritoneal cavity of bear- (100 m l/well, 1 : 1000 ﬁnal dilution) for 2 h at room tempera-ing-mice, counted and implanted subcutaneously into right ture. The plates were subsequently washed three times withhind groin of experimental mice (2ϫ106 cell/500 m l). One PBS-tween. The reaction was developed by the addition ofday after inoculation, biﬂorin alone (25, 50 mg/kg), 5-FU orthophenylenediamine followed by incubation for 20 min atalone (10 or 25 mg/kg) or biﬂorin (25 mg/kg) plus 5-FU 37 °C. The intensity of the resulting color was read at(10 mg/kg) were dissolved in DMSO 10% and administered 450 nm.intraperitoneally for 7 d. The negative control was injected Statistical Analysis Data are presented as meanϮS.E.M.with DMSO 10%. On day 8, the mice were sacriﬁced. Tu- from n experiments. The differences between experimentalmors, livers, spleens and kidney were extirpated, weighed groups were compared by ANOVA followed by Studentand ﬁxed in formaldehyde 10%. Inhibition ratio (%) was cal- Newman Keuls (pϽ0.05).culated by following formula: inhibition ratio (%)ϭ[(AϪB)/A]ϫ100, where A is average tumor weight of the RESULTSnegative control, and B is that of the treated group. Histopathology and Morphological Observations Effects of biﬂorin on mice transplanted with sarcoma 180After the dissection, tumors, livers, spleens and kidneys were and Ehrlich carcinoma tumors are presented in Tables 1 andﬁxed in formaldehyde 10%, and examined grossly for size, 2. It was observed a signiﬁcant reduction of tumor weight incolor and hemorrhage. A portion of the tumor, liver, spleen biﬂorin treated animals at both doses and both tumor modelsand kidneys were cut into small pieces and, later, the histo- (pϽ0.05).logical sections (5 m m) were prepared and stained with Histopathological analysis of the tumors extirpated fromhematoxylin and eosin. Histological analyses were performed sarcoma 180 and Ehrlich carcinoma control mice showedunder light microscopy. groups of large, round and polygonal cells, with pleomorphic Ki67 Immunohistochemical Detection The following shapes, hyperchromatic nuclei and binucleation (Fig. 2). Sev-method was described previously by our group.13,14) Tumor eral degrees of cellular and nuclear pleomorphism were seen.sections were deparafﬁnized with xylene and dehydrated Mitosis, muscle invasion and coagulation necrosis were alsowith ethanol. The slides were then immersed in water for noticed. In the tumors extirpated from animals treated with10 min. For antigen retrieval, the slides were boiled in citrate biﬂorin at 50 mg/kg/d, 5-FU at 25 mg/kg/d or the associationbuffer (pH 6.0) for 15 min in a microwave and subsequently of biﬂorin (25 mg/kg/d) plus 5-FU (10 mg/kg/d), extensivecooled for 20 min. The slides were then washed in TBS, and areas of coagulative necrosis were observed, while in thethe endogenous peroxidases were blocked by 0.3% hydrogen groups treated with biﬂorin (25 mg/kg/d) or (5-FU 10peroxide for 15 min. After washing with TBS, the sections mg/kg/d) there was only a discreet increase of the areas withwere incubated overnight at 4 °C with mouse antibodies for coagulative necrosis.Ki-67 at the concentration of 1 : 50. After 24 h, the slides Ki67 staining for cell proliferation was performed on sar-were washed and incubated with a multilink antibody for coma 180 tumors removed on day 8 from the untreated ani-20 min, washed in TBS and incubated for 20 min with mals and treated with 5-FU (10 mg/kg/d), biﬂorin (25,avidin–biotin-peroxidase complex. After washing with TBS, 50 mg/kg/d) or the association of biﬂorin (25 mg/kg/d) withthe slides were incubated for 3 min with diaminobenzidine, 5-FU (10 mg/kg/d). Nuclear staining and good preservationand ﬁnally, counter-stained with hematoxylin prior to mount- of morphological details were observed in all tumors sectionsing. The percentage of proliferating neoplastic cells was immunostained with Ki67 antibody. Figure 3 shows theevaluated directly under optical microscope. To quantify the amount of Ki67 positive cells in analyzed slides. Resultsamount of proliferation, all Ki67-positive cells were counted from this analysis show that the relative number of Ki67-pos-in 6 random ﬁelds per slide. itive tumor cells was substantially smaller in tumors from Subcutaneous Immunization Two groups of 10 swiss mice treated with 5-FU and biﬂorin at both doses and withmice (provided by the Central Animal House of Universi- the association (biﬂorinϩ5-FU) than in control tumorsdade Federal do Ceará) had been immunized subcutaneously (pϽ0.05).
1418 Vol. 30, No. 8Table 1. Inhibition Rate of Compounded Drugs on Mice Transplanted Sarcoma 180 Tumor Liver Spleen Kidney Dose Drug (g/100 g body (g/100 g body (g/100 g body Tumor (g) Inhibition (%) n (mg/kg/d) weight) weight) weight) Control — 5.21Ϯ0.16 0.65Ϯ0.02 1.16Ϯ0.04 1.78Ϯ0.09 — 20 Biﬂorin 25 5.47Ϯ0.19 0.78Ϯ0.03a) 1.25Ϯ0.05 1.51Ϯ0.07 14.89 14 50 5.17Ϯ0.10 0.71Ϯ0.05 1.00Ϯ0.04 0.88Ϯ0.14a) 50.53 10 5-FU 10 5.73Ϯ0.30 0.50Ϯ0.03a) 1.25Ϯ0.06 1.07Ϯ0.10a) 39.84 10 25 5.12Ϯ0.31 0.30Ϯ0.03a,b,c) 1.68Ϯ0.08 0.46Ϯ0.08a) 74.19 13 Biﬂorinϩ5-FU 25ϩ10 4.57Ϯ0.28b) 0.54Ϯ0.04a,b) 1.11Ϯ0.13 0.61Ϯ0.11a,c) 65.83 10 Data are presented as meanϮS.E.M. from n experiments. Signiﬁcant differences were evaluated by ANOVA followed Student Newman Keuls: a) pϽ0.05 compared to controlgroup. b) pϽ0.05 compared to biﬂorin 25 mg/kg/d. c) pϽ0.05 compared to biﬂorin 25 mg/kg/d and 5-FU 10 mg/kg/d independently administered.Fig. 2. Sarcoma 180 Tumors Removed on Day 8 from Animals Treated with: A) DMSO 10%, B) 5-FU (25 mg/kg/d), C) Biﬂorin (50 mg/kg/d), D) Biﬂorin(25 mg/kg/d)ϩ5-FU (10 mg/kg/d) Horizontal barsϭ1 mm. served an increase in liver weight (pϽ0.05, Table 2). 5-FU at 25 mg/kg/d reduced the spleen weight of animals inoculated with both tumors (pϽ0.05, Tables 1, 2). Histopathological analyses of kidneys removed from animals-treated with 5-FU (10, 25 mg/kg/d), biﬂorin (25, 50 mg/kg/d) or the association of biﬂorin and 5-FU showed hydropic degeneration of proximal tubular epithelium but the glomeruli structure was essentially preserved.Fig. 3. Effect of Biﬂorin Alone and in Combination with 10 mg/kg of 5- On the other hand, histopathological analyses of the liverFU on Sarcoma 180 Cell Proliferation Using Ki67 Antibody showed that all treated animals including control ones, that Ki67-positive cells from 4—6 ﬁelds/tumor slide were counted and the meanϮS.E.M. received only vehicle, showed alterations as Kupffer cells hy-of positive cells was calculated. Saline solution was used as the negative control.10 mg/kg/d of 5-FU was used as the positive control. ∗ pϽ0.05, ANOVA followed by perplasia, intense ballooning degeneration of hepatocytesStudent Newman Keuls. and portal tracts and centriolobular vennus congestion. Fur- ther than these alterations, animals treated with biﬂorin 50 mg/kg/d, 5-FU (10, 25 mg/kg/d) or biﬂorin 25 mg/kg/dϩ After treatment with biﬂorin (25 mg/kg/d), the spleen 5-FU 10 mg/kg/d presented also steatosis microvesicular, si-weight of mice inoculated with sarcoma 180 was sig- nusoidal hemorrhage and focal inﬁltrated of chronic inﬂam-niﬁcantly increased (pϽ0.05), while there were no differ- matory cells (Fig. 4). In the spleen, biﬂorin-treated miceences for liver and kidney (Table 1). At the highest dose showed a discreet increase of the white pulp and nest of(50 mg/kg/d), this augment was not observed. On the other megakaryocytic, which suggests an immunomodulatory ac-hand, in mice inoculated with Ehrlich carcinoma, spleen tivity (Fig. 5).weight was increased only after the treatment with biﬂorin at To investigate the effect of biﬂorin on the induction of hu-50 mg/kg/d ( pϽ0.05, Table 2). In this group, it was also ob- moral immune response of mice immunized with OVA, the
August 2007 1419Table 2. Inhibition Rate of Compounded Drugs on Mice Transplanted Ehrlich Tumor Liver Spleen Kidney Dose Drug (g/100 g body (g/100 g body (g/100 g body Tumor (g) Inhibition (%) n (mg/kg/d) weight) weight) weight) Control — 5.04Ϯ0.18 0.78Ϯ0.06 1.14Ϯ0.03 1.89Ϯ0.68 — 22 Biﬂorin 25 4.99Ϯ0.20 0.69Ϯ0.05 0.99Ϯ0.05 1.99Ϯ0.38 12.29 7 50 5.92Ϯ0.15a,c,d ) 0.96Ϯ0.07c,d) 1.32Ϯ0.03 1.24Ϯ0.33a) 45.39 9 5-FU 10 4.92Ϯ0.25 0.65Ϯ0.07 1.26Ϯ0.08 1.34Ϯ0.51a) 41.15 7 25 4.09Ϯ0.35 0.23Ϯ0.03a,b,c,d) 1.13Ϯ0.08 0.58Ϯ0.26a) 74.57 9 Biﬂorinϩ5-FU 25ϩ10 4.69Ϯ0.18 0.57Ϯ0.05 1.12Ϯ0.03 0.47Ϯ0.19a,c) 79.36 9 Data are presented as meanϮS.E.M. from n experiments. Signiﬁcant differences were evaluated by ANOVA followed Student Newman Keuls. a) pϽ0.05 compared to controlgroup. b) pϽ0.05 compared to biﬂorin 25 mg/kg/d. c) pϽ0.05 compared to biﬂorin 25 mg/kg/d and 5-FU 10 mg/kg/d independently administered. d) pϽ0.05 compared to biﬂorin25 mg/kg/dϩ5-FU 10 mg/kg/d.Fig. 4. Liver of the Animals Treated with: A) DMSO 10%, B) 5-FU (25 mg/kg/d), C) Biﬂorin (50 mg/kg/d), D) Biﬂorin (25 mg/kg/d)ϩ5-FU (10 mg/kg/d) Black arrows show Kupffer cells hyperplasia. Horizontal barsϭ1 mm.OVA-speciﬁc antibody levels in the sera were measured prior tumor growth. On the other hand, all tested compounds, in-to immunization and at 7, 14 and 21 d after, at the dilution of cluding lapachol, were active against Ehrlich carcinoma.1 : 40 by ELISA. Results are shown in Fig. 6. The amount of b -Lapachone was active against sarcoma 180 cells in vitroOVA-speciﬁc total Ig in the sera was signiﬁcantly increased and Yoshida sarcoma and Walker 256 carcinosarcoma inby biﬂorin at a dose of 1.0 mg (40 mg/kg) compared with vivo.22,23) Biﬂorin, on the other hand, presented activityOVA control (pϽ0.05). against both tumors with a very similar proﬁle. In addition to the antitumor activity observed for biﬂorinDISCUSSION alone, this compound was also able to increase the response elicited by 5-FU in mice inoculated with both sarcoma 180 The present work reports the antitumor activity of biﬂorin, and Erlich carcinoma. This is a very interesting ﬁnding sincean o-naphthoquinone isolated from C. biﬂora, on mice trans- one option to improve the efﬁcacy of anticancer therapy is toplanted with sarcoma 180 and Erhlich carcinoma. These develop optimal combination regimens of chemotherapeuticmodels are mouse-originated tumors frequently used in anti- drugs, which may also reduce the side effects of the treat-tumor related research in vivo.15,16) In fact, the naphtho- ment.quinones, mainly represented by b -lapachone, constitutes Immunohistochemical staining of cells for proliferation-a promising group of compounds with antitumor prop- associated proteins offers information about the tumor’s pro-erties.17—20,21) Lima et al. (1972) had studied the antitumor liferation rate. The monoclonal antibody Ki67, described byactivity of naphthoquinones, e.g. juglone, lapachol, lawsone Gerdes et al. (1983),24) is a mouse monoclonal antibody thatand plumbagine, on the same experimental models as the identiﬁes a nuclear antigen associated with G1, S, G2 and Mpresent paper, showing that these compounds, with the ex- phases. This molecule is expressed along the entire cellception of lapachol, were able to inhibit the sarcoma 180 cycle, except on G0 and early G1 phases.25) Thus, results ob-
1420 Vol. 30, No. 8Fig. 5. Spleen of the Animals Treated with: A) DMSO 10%, B) 5-FU (10 mg/kg/d), C, D) Biﬂorin (25 mg/kg/d), E, F) Biﬂorin (25 mg/kg/d)ϩ5-FU(10 mg/kg/d) Horizontal barsϭ1 mm. ber (2002)26) discussed the relation between tumor growth, cell proliferation and Ki67 staining, concluding that Ki67 staining analysis using tumor microsections do not necessar- ily represent a direct relationship with tumor size. Moreover, this marker is better related to tumor aggressiveness and ma- lignancy.27) Nevertheless, present data suggested that biﬂorin altered tumor behavior. In a previous work, we demonstrated that in vitro antitu- mor properties of biﬂorin may differ from that described for b -lapachone and lapachol, since biﬂorin displayed antioxi-Fig. 6. Effect of Biﬂorin (1 mg) on OVA-Speciﬁc Total Ig Antibody dant effects instead of prooxidant, as observed for these other Mice were immunized subcutaneously with OVA (50 m g, ᭹) or with OVA naphthoquinones.12,20,28) In fact, the mechanisms of cell death(50 m g)ϩbiﬂorin (1 mg, ). Sera were collected prior to immunization and 7, 14 and triggered by b -lapachone remains controversial.18) Despite21 d after immunization. Antibodies were detected by ELISA at the dilution of 1 : 40. the induction of oxidative stress, b -lapachone inhibited theData are presented as meanϮS.E.M. from 10 animals. ∗ pϽ0.05, ANOVA followed byStudent Newman Keuls. catalytic activity of topoisomerase I29) and selectively in- duced apoptosis in transformed cells but not in proliferatingtained with Ki67 staining in sarcoma 180 tumor reinforces normal cells.19)that the activity of biﬂorin was related to a reduction in the Biﬂorin mechanisms of tumor cell death are unknown andtumor proliferation rate. In fact, it was already reported that preliminary data suggests that this compound could triggerbiﬂorin shows a cytotoxic effect towards tumor cell lines in leukemia cell differentiation in vitro. Present data shows thatvitro.12) It is worthwhile to mention that the reduction in Ki67 biﬂorin signiﬁcantly increased the serum antibody produc-staining was signiﬁcant even in tumors removed from animal tion in mice immunized with OVA, which indicates an im-treated with biﬂorin at the dose of 25 mg/kg/d, where the munostimulant activity for this natural naphthoquinone. Fur-tumor weight was not signiﬁcantly reduced. Ruban and Far- ther, in mice treated with biﬂorin, it was observed morpho-
August 2007 1421logical alterations in spleen compatible also with an im- 10) Fonseca A. M., Pessoa O. D. L., Silveira E. R., Lemos T. L. G., Montemunostimulant activity.30) F. J. Q., Braz-Filho R., Mag. Reson. Chem., 41, 1038—1040 (2003). 11) Lima G. O., D’albuquerque I. L., Magalhães Neto B., Albuquerque M. The histopathological analyses of organs removed from M., Rev. Inst. Antibiot. Recife., 1, 95—98 (1958).treated animals suggest that biﬂorin possesses only a weak 12) Vasconcellos M. C., Montenegro R. C., Militão G. C. G., Pessoa O. D.,toxicity, since most of the observed morphological alter- Fonseca A. M., Lemos T. G. L., Pessoa C., Moraes M. O., Costa-ations in biﬂorin-treated animals were also seen in the con- Lotufo L. V Z. Naturforsch., 60c, 394—398 (2005). .,trol group, suggesting that these effects could be related to 13) Magalhães H. I. F., Veras M. L., Torres M. R., Alves A. P. N. N., Pes- soa O. D. L., Silveira E. R., Costa-Lotufo L. V Moraes M. O., Pessoa .,hepatocytes metabolism.31,32) Nevertheless, some alterations C., J. Pharm. Pharmacol., 58, 235—241 (2006).like microvesicular steatosis accompanied by ballooning de- 14) Bezerra D. P., Castro F. O., Alves A. P. N. N., Pessoa C., Moraes M. O.,generation of hepatocytes were observed in biﬂorin and 5-FU Silveira E. R., Lima M. A. S., Elmiro F. J. M., Costa-Lotufo L. V.,treated animals, but these effects, according to the literature, Braz. J. Med. Biol. Res., 39, 801—807 (2006). 15) Ito H., Shimura K., Itoh H., Kawade M., Anticancer Res., 17, 277—could also be related to a weak hepatotoxicity.31,33,34) Re- 284 (1987).moval of drugs or dosage adjustment usually leads to rapid 16) Lee Y. L., Kim H. J., Lee M. S., Kim J. M., Han J. S., Hong E. K.,improvement.31) The liver is an organ with great adaptive and Kwon M. S., Lee M. J., Exp. Anim., 52, 371—375 (2003).regeneration abilities. For example, the increase in endoplas- 17) Li C. J., Li Y. Z., Pinto A. V Pardee A. B., Curr. Cancer Drug .,mic reticulum produced by long-term treatment with anticon- Targets, 96, 13369—13374 (1999). 18) Li Y., Sun X., LaMont T., Pardee A. B., Li C. J., Proc. Natl. Acad. Sci.vulsant drugs is commonly regarded as an adaptive phenom- U.S.A., 100, 2674—2678 (2002).enon. On the other hand, regeneration of hepatic tissues oc- 19) Kumi-Diaka J., Saddler-Shawnette S., Aller A., Brown J., Cancer Cellcurs in many diseases, except in the most deleterious ones. Int., 4, 5 (2004).Even when the hepatocellular necrosis is present, but the 20) Reinicke K. E., Bey E. A., Sentle M. S., Pink J. J., Ingalls S. T., Hop-conjunctive tissue is preserved, the regeneration is merely pel C. L., Misico R. I., Azarc G. M., Burton G., Bornamann W. G., Sutton D., Gao J., Boothman D. A., Clin. Cancer Res., 11, 3055—complete.31,33) Moreover, the kidney alterations observed 3064 (2005).in biﬂorin treated animals could also be considered dis- 21) Lima O. G., Maciel G. M., Oliveira L. L., Lacerda A. L., Moreira L.crete.34—36) C., Martins D. G., Rev. Inst. Antibiot. Recife., 12, 3—12 (1972). In conclusion, biﬂorin exhibited antitumor effects on ex- 22) Santana C. F., Lima O. G., D’Albuquerque L., Lacerda A. L., Martinsperimental tumors without an expressive toxicity. This activ- D. G., Rev. Inst. Antibiot. Recife., 8, 89—94 (1968). 23) Do Campo R., Cruz F. S., Boveris A., Muniz R. P., Esquivel D. M.,ity seemed to be related to its immunestimulant properties Biochem. Pharmacol., 28, 723—728 (1979).and to the reduction of tumor proliferation rate. Moreover, 24) Gerdes J., Schwab U., Lemke H., Stein H., Int. J. Cancer, 31, 13—20despite the low potency observed for the use of biﬂorin (1983).alone, it increased the efﬁcacy of 5-FU. Further studies are in 25) Gerdes J., Lemke H., Baisch H., Wacker H. H., Schwab U., Stein H., J. Immunol., 133, 1710—1715 (1984).progress to determine its mechanism of action. 26) Rubin E., Farber J. 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